This study is concerned with two aspects of the immune response. The first relates to the affinity limitations inherent in the expression of the germ-line immunoglobin gene segments and its significance for the affinity restriction of IgM antibody. The second aspect is directed to the specificity and affinity implications of the differences between immunogenic peptides in the free state i.e. highly flexible, and in the constrained state in proteins. Both projects will utilize hybridomas producing monoclonal antibody to a defined epitope. There are now available 60 cell lines producing anti-dansyl (5-dimethylaminoaphthalene-1-sulfonate) IgM and IgG antibodies whose binding constants cover a 100,000-fold range. From these cell lines mRNA will be isolated and the cDNA prepared by the primer extension method. Nucleotide sequencing of the variable regions will be carried out by the Maxam-Gilbert procedure using this cDNA or by the dideoxy method with the mRNA and the above primer. For the studies with peptides hybridomas producing monoclonal antibodies against synthetic peptides whose sequences are present on the surface of well-defined proteins will be prepared. Specifically purified antibody will be obtained from ascites and used in affinity measurements. The synthetic peptides will be conjugated to fluorescent probes and fluorescence methodology employed for the determination of affinity. Competitive binding experiments will allow the determination of the binding constants for the peptides present in a protein.